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  • C12N2830/00—Vector systems having a special element relevant for transcription
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
  • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

• the present invention relates to the use of retroviral vectors, and especially lentiviral vectors for the preparation of compositions which are capable of inducing or contributing to the occurrence or improvement of an immunogical reaction in vitro, and in a preferred embodiment in vivo, against epitopes which are encoded by nucleotide sequences present in the vectors.
• vectors prepared in accordance with the present invention enable to obtain a cell-mediated immune response, especially a Cytotoxic T Lymphocytes (CTL) reaction against epitopes.
• CTL Cytotoxic T Lymphocytes
• the invention provides means which could be used in treatment protocols against tumors and cancer and especially could be used in protocols for immunotherapy or vaccination therapy against tumors.
• the invention also discloses means that could be used for the treatment or prophylaxis of infectious diseases, especially diseases associated with virus infection and for instance, with retrovirus infection.
• the cell-mediated immune response and especially the CTL response associated with the treatment by the compositions of the invention can be specific for the antigen of the tumor or of the virus or virus infected cells, and can also be restricted to specific molecules of the MHC (Major Histocompatibility Complex).
• the invention relates to the use of the vectors in immunogenic compositions, in order to obtain a cell-mediated immune response restricted to Class I molecules of the MHC complex and for instance restricted to HLA-A2 or B7 molecules.
• the invention relates to an immunogenic composition comprising a recombinant vector which comprises a polynucleotide encompassing the cis-acting central initiation region (cPPT) and the cis-acting termination region (CTS).
• cPPT central initiation region
• CTS cis-acting termination region
• the DNA triplex stimulates the nuclear import of vector DNA genomes, these regions being of retroviral or retroviral-like origin, said vector comprising in addition a defined nucleotide sequence (transgene or sequence of interest) and regulatory signals of retrotranscription, expression and encapsidation of retroviral or retroviral-like origin, wherein the composition is capable of inducing or of stimulating a CTL (Cytotoxic T Lymphocytes) or a CD4 response against one or several epitopes encoded by the transgene sequence present in the vector.
• CTL Cytotoxic T Lymphocytes
• the cell-mediated immune response and especially the CTL response or the CD4 response against one or several epitopes is a memory CTL or a CD4 response.
• the presence, in the vector, of the cPPT and CTS regions enables the triplex DNA structure to be formed thereby influencing and especially improving the nuclear import of the genome of the vector in cells recombined with said vector.
• This capacity of the immunogenic composition according to the invention to induce, improve or in general be associated with the occurrence of a memory CTL response enables to propose the use of the immunogenic composition in protocols of anti-tumor therapy or antivirus or antipathogenic therapy, including when the immune response has to be induced on long lasting period of time or at least inducible when a response is sought at a period of time which can be long term induction of the response, after the administration of the immunogenic composition.
• the immunogenic composition can be used for the preparation of therapeutic composition for the treatment of tumor diseases or infectious diseases, for instance by bacteria or viruses, by inducing or stimulating or participating to the occurrence of a cell-mediated immune response, especially a CTL response or a memory response.
• the immunogenic composition of the invention as a consequence of the presence of the triplex structure in the sequence of the vector, resulting from the presence of the cPPT and CTS regions in the vector and in the vector particles, enables the stimulation of the nuclear import of the genome of the vector, in target cells.
• the induced epitopes in the vector can be self or non-self.
• the present invention covers also the use of a nucleotidic sequence comprising the cPPT and CTS sequences of retroviral or synthetic origin for increasing the entry of nucleotidic or peptidic sequences in the nucleus of the target cells or recipient cells.
• the said triplex sequence comprises foreign sequences or self sequences with respect to the recipient cells.
• the invention discloses a composition that could be used in therapeutic protocols that present analogies with vaccination protocols, for the treatment of tumors and especially as anti-cancer or anti-infectious diseases treatment.
• this transgene or sequence of interest can be a sequence encoding one or several epitopes of one or several tumor cells, for instance epitopes that have been identified in potential target antigens for the induction of a cell-mediated immune response against the tumor.
• epitopes forming a polyepitope can be encoded by the transgene of the invention.
• they can be derived from target antigens identified in the tumor, and can be chosen in such a way that when their coding sequence is combined to form the transgene, a cell-mediated immune response is obtained against all the epitopes or against most of them.
• the cell- mediated immune response can be assayed in vitro or in a preferred embodiment in vivo. Protocols enabling to carry out such assays are described in the Examples.
• Target antigens have been identified in several types of tumors and in particular in melanomas or in carcinomas, including renal carcinomas, Bladder carcinomas, colon carcinomas, lung carcinomas, breast cancer, leukemia and lymphoma...
• the immunogenic composition can be used in order to obtain a cell-mediated immune response, in infectious diseases, including viral-associated infection, or infection linked to any kind of pathogen, including Mycobacteria, for instance, M tuberculosis.
• antigens capable of eliciting a cell-mediated immune response can be identified and their coding sequences inserted in the vector used in the immunogenic composition.
• the des gene of M tuberculosis can be used
• the vectors which are used in the immunogenic compositions may express epitopes or present on proteins (including glycoproteins or other protein-derived compounds) identified as target antigens on tumor cells or on virus-infected cells.
• epitopes, polypeptides or proteins used to provide epitopes can be modified, for instance by mutation, deletion or insertion and for example can be modified to improve their stability.
• the invention also relates to an immunogenic composition
• an immunogenic composition comprising recombinant retroviral particles comprising :
• transgene a recombinant nucleotide sequence containing a defined nucleotide sequence (transgene), placed under the control of regulatory signals of transcription and expression, regulatory signals of retrotranscription, expression and encapsidation and,
• cPPT central initiation region
• CTS cis-acting termination region
• the DNA fragment encompassing the cPPT and CTS cis-active sequences is able to adopt a three stranded DNA structure the "DNA triplex" after reverse transcription and to stimulate the nuclear entry of the vector DNA.
• the immunogenic composition which is capable of inducing or of stimulating a CTL (Cytotoxic T Lymphocytes) response against one or several epitopes encoded by the transgene sequence present in the vector, comprise recombinant retroviral vector particles comprising: a) a gag polypeptide corresponding to nucleoproteins of a lentivirus or to functional derived polypeptides (GAG polypeptides), b) a poi polypeptide constituted by the RT, PRO, IN proteins of a lentivirus or a functional derived polypeptide (POL polypeptide), c) an envelope polypeptide or functional derived polypeptides (ENV polypeptides), d) a recombinant nucleotide sequence comprising a defined nucleotide sequence (transgene or a sequence of interest), coding for one or several epitopes, placed under the control of regulatory signals of transcription and expression, a sequence containing regulatory signals of retrotranscription, expression
• CTL Cyto
• the recombinant retroviral vector particles which are present in the immunogenic composition replying to one or the other above-definition are in a preferred embodiment capable of inducing, improving or being associated to the occurrence of a memory cell-mediated immune response and especially a memory CTL response.
• the immunogenic composition containing the vectors or vector particles can be prepared in accordance with several possible embodiments.
• the immunogenic composition is prepared in such a way that the sequences of retroviral origin are derived from a lentivirus genome.
• these sequences are of retroviral-like origin and are derived from retrotransposon.
• the transgene or sequence of interest which is contained in the recombinant vector is contained in an expression cassette including regulatory signals of transcription and expression.
• the regulatory signals of retrotranscription, expression and encapsidation of the vector are of lentiviral origin and the polynucleotide comprising the cPPT and CTS regions is of lentiviral origin.
• the regulatory signals of retrotranscription, expression and encapsidation and the polynucleotide comprising the cPPT and CTS regions in the vector are derived from a HIV-type retrovirus, in particular HIV-1 or HIV-2.
• viruses and especially lentiviruses can be used to design the regulatory signals of retrotranscription expression and encapsidation, and also to derive the polynucleotide comprising the cPPT and CTS regions.
• the lentiviruses CAEV, EIAV, VISNA, HIV, SIV or FIV can be used therefore.
• sequences encoding polypeptides or proteins necessary for the transcomplementation of the vectors are for instance GAG, POL and ENV proteins derived from lentiviruses, and especially from HIV, including HIV-1 and HIV-2 retroviruses.
• the GAG and POL sequences may be derived from a different virus than the ENV sequence.
• GAG and POL sequences can be derived from the HIV retrovirus and the ENV sequence can be derived from another virus or retrovirus, and can be either amphotropic or ecotropic ENV sequences.
• the ENV sequence is derived from the vesicular somatitis virus (VSV).
• VSV vesicular somatitis virus
• the immunogenic composition of the invention which comprises the recombinant retroviral-like particules are in a preferred embodiment capable of generating a memory cell-mediated response, especially a memory CTL response, in accordance with the above-disclosed features.
• the invention also relates to vector constructs which have been deposited with the CNCM (Collection Nationale de Culture de Microorganismes at Institut Pasteur in Paris, France) on October 11, 1999.
• CNCM Collection Nationale de Culture de Microorganismes at Institut Pasteur in Paris, France
• a first vector is pTRIP.TEL/AML-IRES-GFP, deposited under number I- 2326 on October 11, 1999 and a second vector is designated pTRIP-ILKE -IRES- GFP, and has been deposited under number I-2327 on October 11 , 1999.
• a third vector, pTRIP.DES-IRES-GFP has been deposited with the CNCM under number 1-2331 on October 11 , 1999.
• sequences encoding the antigens that are present in the above constructs can be replaced by any other antigen or epitope of interest, including the above cited complete DES gene of M tuberculosis.
• the vectors, vector particles and immunogenic compositions comprising the same are designed in such a way that the cPPT and CTS regions are localized centrally within the sequence of the vector.
• « localized centrally » it is meant that the cPPT and CTS regions are in the center of the sequence of the vector, or approximately in the center of this sequence.
• the cPPT and CTS regions can be within the central one third of the retrotranscribed linear vector DNA.
• the central localization of the triplex sequence formed during viral retrotranscription as a consequence of the presence of the cPPT and CTS sequences enable an improvement of the level of transduction of cells contacted with teh vector or vector particles.
• the transcription unit of the vector including the transgene can be inserted within the U3 region of the LTR region. Accordingly, after retrotranscription, the transgene is duplicated and therefore appears on each side of the triplex sequence, therefore enabling the triplex sequence to be localized at the central position in the vector, whatever the size of the transgene.
• the invention also relates to cells which have been put in contact with the immunogenic composition according to the invention and especially relates to recombinant cells transduced by the vector or vector particles of the immunogenic composition.
• These cells are advantageously antigen presenting cells.
• these cells can be chosen among lung cells, brain cells, epithelial cells, astrocytes, mycroglia, oligodendrocyt.es, neurons, muscle, hepatic, dendritic, neuronal cells, cells strains of the bone marrow, macrophages, fibroblasts, hematopoietic cells.
• the immunogenic composition of the invention can thus be used in treatment protocols or for the preparation of treatment compositions for the therapeutic treatment of tumors and especially of cancer, either to generate a primary cell-mediated immune response, especially a CTL response which is advantageously a memory CTL response.
• a primary cell-mediated immune response especially a CTL response which is advantageously a memory CTL response.
• it can be used as an adjuvant treatment with other known anticancer treatment.
• the immunogenic composition of the invention can be used in association with chemotherapy or immunochemotherapy or other approaches to anticancer treatment.
• anticancer treatment » it is intended in accordance with the present invention, the inhibition of growth of the tumor or potential growth of the tumor or the inhibition of spread of the malignant cells, including the possibility of controlling formation of metastasis, or both.
• the expression « anticancer treatment » relates to protocols that are used to control the malignant growth and spread of tumors, do anticipate recurrence of the disease, especially in view of the fact that the immunogenic composition is capable of inducing or improving, and in general participating to, a memory cell-mediated response.
• Tumors that may be treated with the compositions of the invention, are for instance melanomas or carcinomas including (lung, bladder, renal, colon) and Lymphoproliferation.
• the tumors that may be treated are also all the tumors expressing tumor specific antigens including self protein mutated and/or self protein surexpressed.
• Every possible acceptable ways of administration of the immunogenic composition of the invention are of interest including administration protocols comprising ex vivo steps, for instance ex vivo transduction of target cells followed by administration of the treated cells to the patient to be treated.
• the immunogenic composition according to the invention can be directly administered to the patient through usual routes of administration, including systemic (IV), locally, or cutaneously, intradermic, for instance intratumoral, administration routes.
• routes of administration including systemic (IV), locally, or cutaneously, intradermic, for instance intratumoral, administration routes.
• the immunogenic composition according to the invention can be directly administered to the patient, in such a way that it will induce, improve or participate in vivo to the occurrence of a cell-mediated immune response, especially a CTL-mediated immune response.
• the immunogenic compositions are used so that they can enable the occurrence of a long-term memory cell mediated response.
• the immunogenic composition of the invention has a particular interest due to the property of the cPPT and CTS sequences which are present in the vector and vector particules, to induce or to stimulate the nuclear import of the vector genome in the target cells.
• immunogenic compositions of the invention can be used to elicit or stimulate a cell-mediated immune response against multiple epitopes encoded by the nucleotides sequence of interest or transgene present in the vector or vector particles, and they can also be used to elicit or stimulate a cell-mediated immune response against the product of the entire sequence of a gene, for instance a gene of a pathogenic agent or fragments of said gene capable to encode at least 8 to 15 amino acids preferably 9 to 12 amino acids.
• the invention covers also a nucleotidic sequence comprising nucleotidic sequence encoding a multiple repeat (at least 2 identical sequences) of said amino acid sequence inducing a cellular response and/or an amino acid sequence containing at least 2 different sequences corresponding to 2 epitopes of different pathogens or tumoral antigens.
• Figure 1 HIV1 -derived triplex DNA positive recombinant vector encoding a melanoma polyepitope.
• Figure 3 GFP expression in human cells 5 days after their transduction by the central DNA triplex positive or negative HIV1 -derived vectors.
• Figure 4 In vitro CTL responses using human dendritic cells.
• FIG. 5 Restriction Card of pHR.MEL-IRES-GFP vector Melanome specific CTLs mono or polyepitopical sequences Construction of HR.MEL-IRES-GFP
• HIV specific CTLs mono or polyepitopical sequences, the epitope of which I9V.(ILKE) ; (RT 476-484)
• TRIP.ILKE-IRES-GFP was constaicted by inserting the PCR product of ILKE into TRIP ⁇ E IRES-GFP, between the CMV promotor and the IRES.
• the region surrounding the CTL epitope begining by ILKE was amplified by PCR on the matrix pLAI with the primers :
• ILKE 5TCAGATCTGCCACCATGGCACTAACAGAAGTAATACCAC 3' 3 RIILKE : 5' CGGAATTCTTATTGGCCTTGCCCCTGCTTC 3'.
• a Kozak sequence was inserted into the upstream primer and a stop codon was inserted into the downstream primer.
• the vector expresses a bi-cistronic messenger coding for GFP and a region of the RT gene of VIH, corresponding to a cluster of epitopes, comprising especially the I9V epitope (RT 476-484) restricted to HLA.A2.1 (Walker B.D., 1989 PNAS 86 p. 9514-9518).
• Figure 7 Restriction Map of pTRIP.TELJAML-IRES-GFP vector
• Translocation TEL AML Sequence TRIP.TEL/AML-IRES-GFP was constructed by inserting the PCR product of TEL AML into TRIP ⁇ E IRES-GFP, between the CMV promotor and the IRES. The region surrounding the translocation between TEL and AML was amplified by PCR with the primers :
• a Kozak sequence was inserted into the upstream primer and a stop codon was inserted into the downstream primer.
• Figure 8 Transduction capacity of HlV-derived DNA triplex positive vector encoding 19V epitopic peptide (derived from HIV 1 pol)and GFP of murine dendritic cells that were produced using bone marrow cells from HHD transgenic mice.
• TRIP-des-IRES-GFP vector encoding the DES gene of Mycobacterium tuberculosis.
• Lentiviral vectors have the capacity to transduce cells, including non dividing cells, and are increasingly proposed for gene therapy. Recently, we showed that lentiviral vectors containing the polypurine tract cis-acting sequence (central DNA triplex) exhibit more efficient transduction of human and murine cells than those deleted for the central DNA triplex (Charneau P. et al, J. Mol. Biol. 1994, 241, 651-662). Lentiviral vectors containing or not central DNA triplex and encoding the same HLA-A2.1 restricted melanoma CTL polyepitope have now been tested for their capacity to induce CTL responses.
• the Lentiviridae subclass of retrovirus can infect most cell types including non- dividing cells. This property makes lentivirus attractive for gene therapy.
• Several replication-defective recombinant lentiviral vectors have already been constructed by different groups (Naldini PNAS 93, 11382-8, Science, 1996). These reengineered and detoxified lentiviral vectors are proposed as the most efficient and safe gene therapy vectors (Zufferey R, & Kim V.N. J Virol, 72, 9873-80, 1998).
• H ⁇ V human lentiviral
• VSVG vesicular stomatitis virus G glycoprotein
• the HHD «HLA-A2.1 pure» transgenic mouse (Pascolo et al., J. Exp. Med., 1997, 185 : 2043-2051) allow for an experimentally controlled evaluation of the immunogenic potential of epitopic peptides and of various immunisation strategies.
• HHD mice the capacity of a melanoma polyepitope encoded by different recombinant vectors to induce simultaneous CTL responses within a single animal has been reported.
• the capacity of lentiviral vectors containing or not central DNA triplex and encoding the same melanoma polyepitope for in vivo CTL induction in HHD transgenic mice has first been studied.
• a lentivirus vector encoding the same melanoma polyepitopic motive (Fig. 1 and Table 1) was tested.
• a TRIP-mel-IRES-GFP vector (CNCM 1-2185 deposited on April 20,1999) was administered at 1.25 ⁇ g/p24 per mouse either intravenously, intra peritoneally, or subcutaneously. At least 3 mice per group were used.
• CTL responses were simultaneously induced against most of the ten melanoma epitopes. Similar CTL responses were observed regardless of the route of administration against both peptide loaded (Table 2) and TRIP-mel-IRES- GFP transduced, HHD-transfected HeLa cells (data not shown). However, intraperitoneal injection induced slightly better CTL responses. Strong responses were elicited against NA17-A.nt38 and gp 100.154 epitopic peptides. Significant responses were also observed against gp100.457, MART.1.27, Mage-3, and Tyrosinase.368-D. CTL responses were weak against gp100.209, gp100.280, MART-1.32, and Tyrosinase.1.
• the minimal lentiviral vector dose eliciting a significant CTL response was determined in HHD mice using intraperitoneal injections of TRIP-mel-IRES-GFP vector at six different doses using 4 mice per dose. Two experiments were performed in which effector cells of two mice were mixed to have similar E/T ratios just before the 51 Cr test. In most experiments CTL responses were tested against all melanoma epitopic peptides. Because the results were highly similar and very homogeneous, only CTL responses against NA17/A epitopic peptide were taken into account to compare « dose-effect » relationship for the sake of clarity and simplicity. The best CTL responses were obtained using doses between 500ng and 2500ng/p24 per mouse (Table 2).
• mice Eight mice were injected with the TRIP-mel-IRES-GFP vector. The mice were sacrified either 12 days or 5 months after immunization. After 5 days in vitro stimulation with the melanoma epitopic peptides and two additional day with 10% TCGF, effector cells of four mice were mixed and tested against peptide loaded HHD-transfected RMAS cells. Specific CTL responses were evidenced for all melanoma epitopic peptides but for gp100.209 and Marti .32 in mice immunized 12 days before. Five months after injection of the TRIP-mel-IRES-GFP, all of the primary CTL inducer epitopes still induced strong CTL responses (Fig. 2).
• mice The level of CTL responses 12 days or 5 months after immunization of mice was suprisingly comparable. This suggests that in vivo transduced cells by the lentiviral vector are not distroyed by the immune systeme and continue to produce the encoding melanoma poliepitope.
• HHD mice were immunised individually at the same time with the TRIP-mel-IRES- GFP and HR-mel-IRES-GFP vectors administered intraperitoneally at doses of 800ng, 200ng, 50ng, 12ng, and 3ng/p24 per mouse. At least four mice were tested for each dose in two seperate experiments. After in vitro stimulation by synthetic peptides, the cytolytic capacity of spleen cells was tested using peptide pulsed RMAS-HHD target cells. Because the results were highly homogeneous, only CTL responses against NA17/A, Mart-1.27, gp100.154, and Tyrosinase.368-D epitopic peptide were tested for the sake of clarity and simplicity.
• mice immunised with the TRIP-LV vector at doses of 800ng, 200ng, and 50ng/p24 per mouse elicited better CTL responses than the mice immunised with the HR-LV vector.
• tumor cells such as MT4 and HeLa cells can be transduced up to 30-fold more efficiently by the lentiviral vector containing the central DNA triplex than by the vector lacking the central DNA triplex.
• the transduction capacity of these two lentiviral vectors at different concentrations was then tested on DC from healthy donors or from HHD mice.
• the percentage of DC expressing GFP and their mean intensity of fluorescence were measured by FACS and considered as the transduction level of DC by the two lentiviral vectors.
• the TRIP-GFP vector transduced the cells more efficiently than the HR-GFP vector at all vector concentrations.
• the GFP expression of murine and human DC transduced by the TRIP-GFP vector was respectively up 3- and 7-fold higher than the expression of those transduced by the HR-GFP vector (Fig. 4).
• MNC Mononuclear cells obtained from healthy HLA-A2.1 donors were stimulated in vitro once a week using the DC from the same donor transduced by TRIP mel- IRES-GFP. The presence of GFP expression in the dendritic cells was analysed by FACS to verify efficient transduction. After three weeks, the cytotoxic capacity of the MNC was tested in a 51 Cr assay using peptide pulsed T2 cells as targets in FCS free culture condition.
• the hDC transduced with the TRIP-mel-IRES-GFP vector iduced significant CTL responses against all melanoma epitopic peptides but Marti .31 , whereas non transduced hDC induced only habitual backround responses (Fig. 5).
• lentiviral-derived vectors containing the central DNA triplex induce stronger CTL responses in vivo in HHD mice than those not containing the central DNA triplex. Furthermore, lentiviral vector containing the central DNA triplex can easly transduced hDC in vitro which could be subsequently used for clinical immunotherapy.
• Vector plasmids pTRIP-EGFP derived from HR'CMVLacZ (Naldini et al, PNAS 93,11382-8, 1996). LacZ reporter gene was replaced by the EGFP gene (Clontech). In TRIP-EGFP, the EGFP gene was inserted in the Clal site of a central fragment of HIV-1 LAI comprising cPPT and CTS sequences. EGFP gene was amplified by PCR using Pfu polymerase (Stratagene) from pEGFP-N1 plasmid, adding BamHI and Xhol restriction sites in 5' and 3' respectively. PCR primers were as foolowing: Bam GFP 5' CC GGATCC CCA CCG GTC GCC ACC 3'
• HR GFP vector was constructed by cloning back this PCR fragment into BamHI and Xhol sites of pHR'CMVLacZ, replacing the LacZ ORF by EGFP.
• GFP plasmid vector and TRIPinv GFP in the reverse orientation.
• the same triplex fragment was amplified from pcPPT-AG, pcPPT-D, pcPPT-225 and pCTS plasmids to generate vectors including the same mutations in the cPPT or in the CTS as the corresponding viruses.
• a CTL polyepitope melanoma (mel) fragment was generated by PCR on the pBS mel poly, inserting a kozac consensus sequence inside the primers 5BglMlu Mel:
• 3RIMel 5'CGGAATTCGACCTAAACGCAACGGATG3'.
• the mel PCR fragment was digested by Bglll/EcoRI and inserted in the
• TRIP ⁇ E IRES GFP BamHI/EcoRI sites of TRIP ⁇ E IRES GFP, creating the TRIP ⁇ E mel IRES GFP also called TRIP mel IRES GFP.
• the HR mel IRES GFP was created by exchanging the Ndel/Xhol fragment containing the melanoma polyepitope and the IRES GFP of TRIP mel IRES GFP with that of HR GFP.
• the Ndel site is situated at the end of the CMV promotor.
• the lentiviral vectors were produced as described previously (Naldini I.M. PNAS 1996 and science 1996) by a transient tree-plasmid transfection of 293T cells using the phosphate-calcium technique. Briefly, 293T cells were transfected with 20 ⁇ g of the VSV envelope plasmid (pMDG) and 40 ⁇ g of the various packaging (8.2 or 8.91) and lentiviral vector plasmids. Conditioned media were collected 60h and 84h after transfection. The virus was then concentrated and dNTPs were treated as previously described (Naldini science 1996). Viral titres on HeLa P4.2 cells and MT4 cells were determined by serial dilution and p24 ELISA assay (Naldini Science 1996).
• HeLa cells were infected in triplicate with equivalent amount of vector particules (5 ng P24 per well).
• medium was replaced by 200 ⁇ l of TNB (Tris 50 mM pH 7.5, NaCl 150 mM) and fluorescence of living cells quantitated using a microplate fluorimeter (Victor 2 , Wallac) and EGFP adapted filters (excitation: 485 nm, emission: 520 nm).
• TNB Tris 50 mM pH 7.5, NaCl 150 mM
• fluorescence of living cells quantitated using a microplate fluorimeter (Victor 2 , Wallac) and EGFP adapted filters (excitation: 485 nm, emission: 520 nm).
• Mice. HHD mice have been described previously (Pascolo, 1997).
• mice express a transgenic monochain histocompatibility class I molecule in which the C terminus of the human b2m is covalently linked by a peptidic arm (GGGGS) x 3 to the N terminus of a chimerical heavy chain (HLA-A2.1 a1-a2, H-2D D a3 - transmembrane, and intracytoplasmic domains).
• the H-2D D and mouse b2m genes of these mice have been further disrupted by homologous recombination resulting in complete lack of serologically detectable cell surface expression of mouse histocompatibility class I molecules.
• Human dendritic cells were obtained from cytapheresis products of healthy donors of HLA-A2.1 haplotype (IDM, Paris, France). FACS analysis of these DC using mAbs against CD3, CD14, CD80, CD83, HLA-ABC, and HLA-DR showed immature DC phenotype.
• the hDC were transduced in 1 ml AMV-5 culture medium with the lentiviral vectors at concentrations of 600ng, 300ng, 150ng, and 150ng/p24 per 1.10 ⁇ cells for ten days. The percentage and mean fluorescence intensity of GFP expression in hDC transduced with the two lentiviral vectors were measured by FACS (Becton Dickinson, BD, USA).
• MNC Mononuclear cells from the same donor were stimulated in vitro by the hDC or transduced hDC with a ratio of 4 MNC to 1 hDC.
• the MNC were restimulated twice using the same cryopreserved-transduced hDC and then tested for cytolytic activity in a 4 h 51 Cr-release assay, using as targets T2 cells loaded with relevant or negative control (lnf.m.58) peptides (10 ⁇ g/ml, 5.10 6 cells/ml, in FCS-free RPMI medium, 2 h at RT).
• Bone marrow-derived dendritic cells were generated as previously described [43, 51]. Bone marrow mononuclear cells were cultured in RPMI supplemented with 10% FCS, 2 mM L glutamine, 50 U/ml penicillin, 50 ⁇ g/ml streptomycin, 5.10 5 M 2- mercaptoethanol (complete RPMI medium), further supplemented with 20 ng/ml recombinant mouse GM-CSF and 100 ng/ml recombinant mouse IL4 (both from GENZYME, Cambridge, MA).
• HHD mice were injected either intraperitoneally, intraveneously or subcutaneously with lentiviral vectors for 12 days. Spleen cells from primed mice were then individually restimulated in complete RPMI medium by each epitopic peptide for seven days. The last two days, the cultured cells were restimulated by 10% TCGF. On day 7, cultured cells were tested for cytolytic activity as already described (Pascolo, 1997), in a 4 h 51 Cr-release assay, using as targets HHD-transfected TAP- RMA-S cells loaded with relevant or negative control (lnf.m.58) peptides (10 ⁇ g/ml, 5.10 6 cells/ml, in FCS-free RPMI medium, 2 h at RT).
• the TRIP-mel-ITES- GFP transduced or non transduced HHD-transfected HeLa cells were parallely used as target cells.
• the percentage of specific lysis was calculated as follows: (experimental release - spontaneous release)/(total release - spontaneous release) x 100.
• Example II Evaluation of CTL responses in HHD mice after immunization with the TRIP-des-IRES-GFP vector encoding the DES gene of Mycobacterium tuberculosis.
• the DES gene is disclosed in WO 98/04711.
• the TRIP-des-IRES-GFP vectors were used to transduce HeLa-HDD cells. These cells were transduced and cloned by limiting dilution. The clone expressing the higher level of GFP was selected to use as target cells in classical 51 CrCTL tests.
• HDD mice were injected intraperitoneously using 1.2 micg/p24 per mouse of the TRIP-des-IRES-GFP vector particles.
• Spleen cells of these mice were in vitro stimulated at 12 days post-injection with either 0.2 micg, or 1 micg/p24/ml (2 106 cells per ml) of vector particles or with TRIP-des-IRES-GFP transduced, LPS stimulated syngeneic blast cells with 1 micg/p24/ml/2106 cells/ml.
• cytolytic capacity of cells was tested in a 51 Cr test using des transduced HeLa-HDD target cells.
• Control target cells were HeLa-HDD cells transduced by the melanoma polyepitope (TRIP-mel-IRES-GFP).

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